Primers (prepare by adding 2 uL of each sense and antisense primer + 196 uL water and label as 1 uM primer mix). Design using Stratagene Primer Design Program
dNTP's (2 mM; add 10 uL of each dNTP (100 mM from invitrogen) into 460 uL water, and make 50 uL aliquots)
PFU Turbo
DpnI
Supercompetent XL1-Blue Cells (Stratagene)
Protocol
Mix:
5 uL 10X Reaction Buffer
~20 ng Template (1 uL of minprep)
10 uL of Primer Mix
5 uL of dNTP mix
28 uL of water
Add 1 uL of Pfu Turbo
Run PCR Program (Mutagenesis 8/15/20 minute, depending on template)
95C for 30s
Repeat 18 cycles:
95C for 30s
55C for 1 min
68C for 1 min/kb plasmid length (8 min default, but use 15 or 20 min for larger templates.)
Place on ice for 2 min
Add 1 uL DpnI, mix and spin down. Digest 1h or overnight at 37C.
Thaw XL1-Blue cells on ice and make 50 uL aliquots (round tube)
Add 1 uL of digest to cells and mix.
Incubate 30 min on ice.
Heat at 42 C for 45 s, then place on ice for 2 min.
Add 450 uL SOC preheated at 42C and grow 1h at 37C shaking
Spread out entire transformation on appropriate antibiotic
Grow O/N at 37C
Pick a colony, miniprep and sequence to verify mutation